Water-soluble dyes (e.g., fluorescent, chemiluminescent, visible, and near-IR) are used routinely in molecular biology to label and monitor components of biological reactions. Frequently, residual dyes as well as other organic molecules should be removed before proceeding with many downstream applications. Thus, the present invention is directed to removing dyes and other organic molecules from biological mixtures, particularly in low volume, microfluidic devices.
There is a significant need for high throughput, low volume, integrated microfluidic devices in order to increase sample throughput and reduce the amount of reagents used per sample (thereby reducing cost per sample) in biological reactions. Small volume Polymerase Chain Reaction (PCR) and nucleic acid cycle sequencing reactions are examples of standard molecular biology techniques that are suitable for incorporation into miniaturized formats. In both applications, removal of residual primers, nucleic acid templates, dyes, and other organic molecules are generally necessary prior to any further downstream applications.
One example where such removal methods are used is in the preparation of a finished sample (e.g., purified nucleic acid materials) from a starting sample (e.g., a raw sample such as blood, bacterial lysate, etc.). For example, to obtain a purified sample of the desired materials in high concentrations, the starting sample is typically prepared for PCR after which the PCR process is performed to obtain a desired common PCR reaction product. The common PCR reaction product can then be used in a variety of molecular biological applications, including, for example, sequencing, cloning, genotyping, and forensic applications.
In fluorescence-based DNA sequencing applications, unincorporated dye terminators (i.e., dye-labeled dideoxy terminators such as dideoxynucleotide triphosphates (ddNTPs)) should preferably be removed from the reaction mixture prior to analysis of the DNA sequence fragments. Failure to sufficiently reduce the concentration of dye terminator molecules leads to dye artifacts (i.e., other dye-containing molecules such as dye-labeled dideoxy terminators such as dideoxynucleotide diphosphates (ddNDPs), dideoxynucleotide monophosphates (ddNMPs), and dideoxynucleosides) that can significantly obscure DNA sequence information. Sequencing reaction purification is a desired step in the preparation of samples prior to sequence analysis, particularly when using a capillary electrophoresis (CE) sequencer.
Conventionally, after completion of the PCR or cycle sequencing reaction, the product is generally purified by either alcohol (ethanol or isopropanol) precipitation or gel filtration chromatography. Other protocols using polyalkylene glycol and biotin-streptavidin interactions have also been utilized for sequencing reaction clean-up. Ultrafiltration membranes, phenol/chloroform extraction, and enzymatic treatments are other methods that are commonly used for purification of PCR and sequencing reaction mixtures.
Such conventional technologies for the purification of PCR and nucleic acid sequencing reactions have not proven to be suitable for incorporation into a microfluidic device. Alcohol precipitation utilizes volatile and flammable reagents. Hydrogels (e.g., crosslinked dextrans), commonly used in size exclusion chromatography, require large bed volumes (10× relative the volume of sample) for efficient separation of impurities from product. Gels are first swollen with a relatively large volume of water, centrifuged, and loaded substantially immediately, because, upon dehydration, these materials are prone to cracking. Biotin-streptavidin mediated purifications require the use of custom biotinylated primers for the efficient capture of product. Biotinylated products are generally captured onto streptavidin-treated paramagnetic particles and physically separated from impurities with the use of a magnet. Alternatively, hybridization based purification (HBP) of the PCR or nucleic acid sequencing product can be accomplished by utilizing primers containing specially designed capture tags. Separation of the nucleic acid fragment from the biological matrix can be achieved by hybridization of the capture tag to a complementary strand bound to a solid support. Both the biotin and HBP strategies would require a rinsing step followed by elution of the sequencing or PCR product from the substrate. Although biotin-streptavidin and HBP purification methods yield clean PCR and sequencing fragments, both approaches require customized primers, which can be cumbersome and expensive.
An alternative approach for the removal of residual dye terminators from DNA sequencing reactions involves treating the reaction mixture with an enzyme (e.g., shrimp alkaline phosphatase) to dephosphorylate residual nucleotide triphosphates. Although cleavage of the phosphate groups(s) from the dye-labeled dideoxynucleotide triphosphates alters the mobility of the dye-labeled nucleotides in the sequencing gel, residual dye moieties are not removed from the reaction mixture by this procedure and must still be eliminated prior to injection of the sample into the sequencer. This is generally accomplished by subsequent alcohol precipitation of the digested product.
PCR and sequencing products can also be effectively purified by adsorption of nucleic acid fragments onto beads and silica gel membranes using chaotropic agents. Impurities (e.g., residual primers, dyes, and salts) can be rinsed from the substrate and the purified product eluted. This multi-step bind/rinse/elute purification scheme may also prove to be cumbersome within the context of a microfluidic device.
Yet another method of removing unwanted materials (e.g., dyes) from cycle sequencing (e.g., Sanger cycling) reaction mixtures involves the use of paramagnetic particles. One example of suitable paramagnetic particles incorporating dye terminator removal materials is available under the trade designation RAPXTRACT from Prolinx Inc., Bothell, Wash. Further examples of these materials (and their methods of use) may be found in U.S. patent application Publication No. US 2002/0047003 A1 published on Apr. 25, 2002 and entitled ENHANCED SAMPLE PROCESSING DEVICES SYSTEMS AND METHODS. Unfortunately, however, with such particles, the particles must remain in a hydrated state, which limits the ability to prefabricate particle-loaded devices.
Thus, methods are needed for the removal of dyes and other organic molecules from biological mixtures, such as nucleic acid amplification reaction mixtures (e.g., PCR or cycle sequencing reaction mixtures).